Genegnome imager

Cells seeded in 6-well plates were harvested after two washes with Phosphate buffered saline (PBS) containing phosphatase and protease inhibitors (Thermo Fischer Scientific, Waltham, MA, USA) and lyzed with RIPA lysis Buffer (Sigma-Aldrich, St. Louis, MS, USA). Proteins were separated by 12% SDS-PAGE and transferred onto nitrocellulose membranes [14 (link)]. The membranes were first blocked with 5% milk in TBS-Tween for 1 h, then incubated with appropriate dilutions of primary antibody at 1:1000 for 2 h. Anti-rabbit immunoglobulin-horseradish peroxidase and anti-mouse immunoglobulin-horseradish peroxidase conjugates were used as secondary antibodies (dilution 1:2000, Vectors). The membranes were incubated with Amersham ECL Select or prime Western Blotting Detection Reagent (GE Healthcare, Chicago, IL, USA) and exposed to a film or on an Amersham imager 600 (GE Healthcare) or GeneGnome imager (Syngene, Cambridge, UK). Horseradish peroxidase-conjugated anti-rabbit and anti-mouse antibodies were purchased from Vector Labs. The mouse anti-pan flavivirus envelope E protein mAb 4G2 was produced by RD Biotech. Mouse antibody against HO-1was from Abcam and the mouse antibody against α-tubulin, β-tubulin and M2 FLAG were from Sigma–Aldrich. All Western-blot data are representative of three independent experiments.

Tissues were homogenized (Ultra-Turrax TP, IKA Labortechnik, Wasserburg, Germany) on ice in 300 µl of RIPA lysis buffer (Thermo) per 100 mg of tissue with 1× protease inhibitor cocktail (Thermo) and incubated for 30 min. Lysates were centrifuged at 14 000g, 4°C for 20 min and overall protein concentrations of the supernatant was determined by Pierce BCA Protein Assay (Life Technologies). Samples were incubated at 37°C for 30 min in 1× LDS sample buffer (Life Technologies) and 1× sample reducing agent (Life Technologies), after which 40 µg of protein were loaded per well in a NuPAGE Bis–Tris 4–12% polyacrylamide gel for protein separation via SDS-PAGE electrophoresis. Proteins were transferred to PDVF membrane at 400 mA for 1 h and membrane was blocked for 1 h at 4°C with 5% BSA in TBS + 3% Tween 20. Membranes were subsequently incubated overnight at 4°C with primary antibodies for NPC1 (1:10 000, ab134113, Abcam) and β-tubulin (1:2000, ab6161, Abcam) with 3% BSA in TBS + 3% Tween 20. After 3 washes in TBS, antibody staining was revealed using HRP-conjugated goat anti-rabbit IgG (1:2000, ab6721, Abcam) and goat anti-rat IgG (1:10 000, ab97057, Abcam) incubated for 2 h at RT in TBS + 3% Tween 20 with 3% BSA. Blots were developed with ECL system (SuperSignal West Pico, Life Technologies) and imaged using a Genegnome imager (Syngene, Cambridge, UK).

Cells were washed twice in PBS and lysed with ice-cold RIPA buffer (Santa-Cruz) containing HALT protease/phosphatase cocktail (Pierce/Thermo Scientific). Lysates were incubated on ice for 30 min with gentle, constant agitation. Lysates were centrifuged at 12,000 × g for 20 min at 4 °C to pellet nuclei and other insoluble material. Lysates were treated with Novex sample buffer and heated 10 min at 90 °C. SDS-PAGE was performed on 4–12% Tris-Bis gels (Life Technologies) with MES-based running buffer containing anti-oxidant additive, according to the manufacturer's instructions. Gels were transferred to nitrocellulose (iBlot system, Life technologies). Western blot membranes were blocked with 5% BSA in TBS-T buffer for 1 hour at room temperature, followed by incubation with 5% BSA in TBS-T containing primary antibodies at the indicated concentrations (see Methods, above) overnight at 4 °C with gentle rocking; in the morning, blots were washed in TBS-T and incubated 1 hr at room temperature in 5% BSA in TBS-T containing HRP-conjugated secondary antibodies (1:2500), washed again in TBS-T, and incubated in luminescent peroxidase substrate (ThermoFisher). Imaging of Western blot band luminescence was performed with a GeneGnome imager (Syngene); and band intensities were quantified using Gene Snap software (Syngene).